Protein from Mammalian Cells
Our experience in offering protein expression and cell line development services has translated into extensive expertise in vector design, and in understanding how vectors interact with their hosts to affect the quantity and quality of expressed protein. Below are examples of the approaches we apply as part of our customized protein services to optimize the expression of your protein from mammalian cells.
Common Vector Elements
Recording Open Reading Frame
We have designed and expressed tens of thousands of genes in mammalian cells. By feeding this data into our GeneGPS platform, have identified critical sequence parameters that most strongly affect expression. The graph below shows a strong correlation between the expression of genes predicted by our design algorithm, and the expression level actually observed in HEK cells. This mammalian re-coding enables us to computationally design genes that will express well in the real world.
Signal Sequence
Mammalian cells are frequently used to produce secreted proteins. However, the effectiveness of a mammalian secretion signal sequence is highly dependent on the protein being secreted: signal sequences that work well for one chain of an antibody may be mediocre for another. We have identified many signal sequences and characterized their effectiveness across a range of proteins, including antibody heavy and light chains of different origins.
The antibody-dependence of secretion signal performance is exemplified in the graph below. By using different secretion signals for the heavy and light chains of two different antibodies, we observed a five-fold range in expression levels for Rituxan, and a more than twenty-fold range for Trastuzumab. There is also relatively little correlation between the secretion signals that work well for the two different antibodies. We therefore performed a more extensive study to identify generally good secretion signals.
Effectiveness of secretion signal is highly dependent on protein being secreted
Vector Elements
Promoters, Enhancers, Introns
ATUM uses a variety of viral and cellular promoters as part of our protein expression services. These are combined with introns, which often contain enhancer sequences. Intron processing also enhances mRNA export from the nucleus into the cytoplasm for translation. Many of these elements have been modified from natural sequences during mammalian codon optimization to improve their function.
Different vector elements work better in different cell types
PolyA Tails
Polyadenylation serves to protect the mRNA from degradation, polyA tails also assist in export of mRNA from the nucleus to the cytoplasm. Polyadenylation therefore improves translation, thereby affecting the total amount of functional protein expressed. We have found that the choice of polyA signal sequence has a profound effect on mammalian protein expression.
Choice of polyA signal affects mammalian protein expression
Viral Replication Elements
Internal Ribosome Entry Sites (IRES)
IRES (Internal Ribosome Entry Site) elements create a second, cap-independent site for ribosome binding, resulting in independent expression of two ORFs from a single mRNA molecule. The ratio at which the two proteins are expressed depends on the efficiency of ribosome entry at the IRES relative to ribosome binding 5′ end of the mRNA. As part of our protein expression services or vectorGPS services, we have developed a set of novel IRES elements; each produces a defined expression ratio between the first and second protein in every transfected cell.
Choice of IRES elements to control the ratio of bicistronic expression between two proteins
Stable Expression of Multiple ORFs
Controlling multi-ORF expression
We use our proprietary Leap-In technology for the construction of stable cell lines. Leap-In transposase can integrate expression constructs harboring up to four transcriptional units into a host genome with no rearrangements. By selecting the appropriate vector elements, we can control the relative expression ratios of up to four protein subunits within the same cell.
Leap-In Transposon 2 ORFs
Leap-In Transposon 3 ORFs
Leap-In Transposon 4 ORFs
Selection Markers
A variety of selectable markers are available for conferring resistance to puromycin, neomycin, hygromycin and blasticidin. Alternatively, genes encoding glutamine synthetase and dihydrofolate reductase can be used for drug-free selection.
We have developed several attenuated selectable markers through which selection stringency can be manipulated. Under higher selection stringency, only cells with more copies of the selectable marker, and thus of the expression construct, will survive. For low toxicity molecules (such as monoclonal antibodies), high selection stringency results in highly productive stable cell lines and pools. For challenging or toxic molecules, choosing the appropriate selection stringency enables the creation of stable cell lines optimized for the expression of that molecule.
Transient Vector Performance Varies with Cell Type
DasherGFP Expression in Transiently Transfected HEK293 and CHO Cell Lines
Cell Lines
Individual vector elements, and therefore entire vectors, can behave differently across even closely related cell lines. By understanding the ‘preferences’ of each cell line, we can design the vector most appropriate for that cell line. We have already build vectors optimized for HEK293 and CHO expression hosts, used both in-house at ATUM and also by clients in their proprietary cell lines. In addition to catering for clients working with CHO cell lines, we routinely create new vectors for clients working in non-CHO cell lines with applications in cell therapy such as the Jurkat/T-cell line.
DasherGFP expression in transiently transfected cells. HEK293 (adherent), Expi293® (suspension), CHO-K1 (adherent), Freestyle® CHO-S (suspension), and ExpiCHO® (suspension) cells were transfected at 2 x 106 cells/ml using Lipofectamine 2000; transfections were carried out in triplicate and incubated for 72 hours post-transfection. Cells were lysed using M-PER and expression measured on a fluorimeter. Orange and grey data points are shown for comparison.